It has been known with polyclonal antibodies that the antigen-binding suppresses the hydrogen-exchange of the antibodies even if the antigen is small. In the previous years we have determined the number of the amide protons whose exchange is suppressed by the antigen using three monoclonal antibodies (mAb) to yeast iso-1 cytochrome c and the Fab fragment of one of them. The results have suggested a possibility that the antigen-binding may increase the stability of the hydrophobic cores of the Fab domains which are remote from the antigen combining site. If this hypothesis is correct, the non-covalent interactions in the cores of the Fab domains fragment, particularly those of the variable domains of the heavy and light chains (VH and VL, respectively) and also possibly those of the VH-VL interface, may influence the antigen binding (based on the action-reaction principle). By inspection of the available x-ray structures of the antigen antibody complexes we have realized that the antigen always contracts the particular geometrical site of the antigen combining surface which is related to the VH-VL interface. We call this site as the antigen-combining surface center. We have speculated that the interaction of the antigen with the residues located at the antigen combining surface center might be related to the mechanism in which the antigen binding stabilizes the cores of the Fab domains and therefore important to generate the affinity of the antigen to the antibody. To test this idea we have used mAb 2-96- 12 to yeast iso-1-cytochrome c, one of the above mAbs. In the previous years we have deduced the amino acid sequences of the VH and VL from the cDNAs, constructed molecular models of the VH-VL complex and identified Arg at position 95 of the VH as one of the residues located at the antigen - combining surface center. A baculovirus transfer vector in which the cDNAs encoding the light chain and the VH and the first constant domain of the heavy chain of mAb 2-96-12 were inserted was constructed. Using this vector and the virus DNA the recombinant viruses were generated to express the Fab (wild type) in the insect cells. The Arg 95 yields Lys mutation of the heavy chain was accomplished using oligonucleotide - directed mutagenesis. The expressed wild type and mutant Fabs were purified from the culture media of the virus infected cells and characterized with respect to the immunological activity. Preliminary results appear to show that (a) the bioengineered Fab closely resembles the native mAb with respect to the affinity to the antigen and the fine specificity; (b) the heavy chain Arg 95 yields Lys mutant Fab also exhibits the fine specificity similar to the native mAb; and (c) however, its affinity to the antigen is markedly decreased as compared with the wild type despite the fact that there should be no difference in the charge. Thus, the concept of the antigen combining surface center appears to be useful to identify the residues of mAb which serve as the starting point for the substitution studies of the antigen-antibody interaction before the x-ray structure is determined.